Interferon inducer and process for preparing the same

ABSTRACT

An interferon inducer for preventing the viral infection diseases, prepared from the supernatant liquid of cultured medium, in which has been cultured Bordetella pertusis, or from a fraction obtained by disintegrating the cell body of Bordetella pertusis and then recovering the effective component therefrom.

United States Patent 1191 Nakase et a1.

INTERFERON INDUCER AND PROCESS FOR PREPARING THE SAME Inventors: Yasukiyo Nakase, Yokohama;

Yasuhiko Kojima, Musashino, both of Japan Assignee: The Kitasato Institute, Tokyo, Japan Filed: Mar. 21, 1972 Appl. No.: 236,670

Foreign Application Priority Data Mar. 22, 1971 Japan t. 46-15825 US. Cl 424/115, 195/96, 424/85 Int. Cl C12d 13/00 Field of Search 195/96; 424/85, 115

References Cited UNITED STATES PATENTS 12/1960 Thiece 195/96 Nielsen et a1. 195/96 OTHER PUBLICATIONS ACTA Virologca, Vol. 10, pages 271-272, (1966).

Primary Examiner-Alvin E. Tanenholtz Attorney, Agent, or Firm-Armstrong, Nikaido & Wegner [57] ABSTRACT An interferon inducer for preventing the viral infection diseases, prepared from the supernatant liquid of cultered medium, in which has been cultured Bordetella pertusis, or from a fraction obtained by disintegrating the cell body of Bordetella pertusis and then recovering the effective component therefrom.

4 Claims, No Drawings INTERFERON INDUCER AND PROCESS FOR PREPARING THE SAME This invention relates to an interferon inducer for the viral infection and to a process for preparing the same. More particularly, the invention pertains to an interferon inducer obtained by use of the supernatant of a liquid culture medium, or a liquid containing a disintegrated cell body of, a Phase 111, Phase II or Phase I organism of Bordetella pertusis, [GS Wilson and AA. Miles: Principles of Bacteriology and Immunity, Vol. 1, page 973 (5th ed., 1964), published from Edward Arnold Ltd. (London)] and to a process for preparing the said inducer.

An object of the present invention is to provide a process for preparing an interferon inducer from the supernatant of a liquid culture medium, in which has been cultured a Phase III, Phase II or Phase I organism of Bordelella periiisis.

Another object of the invention is to provide a process for preparing an interferon inducer from a fraction obtained by disintegrating the cell body of a Phase [11, Phase 11 or Phase 1 organism of Bordetella pertirsis and then removing lipopolysaccharide endotoxin and O-antigen from the somatic components of bacteria.

A further object of the invention is to provide an interferon inducer obtained according to any of the above-mentioned processes.

Heretofore, interferon inducers for preventing the viral infectious diseases have been well known according to many research reports such as, for example, Julius S. Youngner: Jour, General Physiology, 56 (1), Part 2, pages 25s-40s (1970); and J. Vilcek: Virology Monograph Vol. 6, Interferon (published from Springer-Verlag, 1969, Wien-New York), particularly pages 21-22. Typical examples of the known interferon inducers include various viruses, bacteria (particularly Gram-negative bacteria), lipopolysaccharide endotoxin of said bacteria, metabolic products of molds, polysaccharides and double stranded-RNA. All these interferon inducers, however, are toxic and, when administered, bring about such side effects as fever and the like, so that they have not been used so frequently.

According to the process of the present invention, it is possible to obtain an interferon inducer which is low toxic and which does not bring about any side efiects. The process of the present invention is carried out ac- A Phase 111, Phase II or Phase I organism of Bardetella pertusis is subjected to shaking culture at 30 to 37C. for 24 to 94 hours by use of a liquid medium such as a modified Cohen-Wheeler culture medium [S.M. Cohen and M.W. Wheeler. Am. J. of Public Health & Nations Health, Vol. 36, pp. 371-376 (1946)]. Thereafter, the supernatant of the cultured medium is recovered by centrifuge and then filtered under sterile conditions, and the filtrate is subjected to ultra-filtration or chroma tography, whereby an effective fraction can be isolated. 2. A cultured cell body of the abovemgntioned Phase III Phase II or Phase I organism of Bordetella pertusis is once disintegrated bysubjecting the cell body for to 30 minutes to physical treatment such as sonic treatment or to mechanical treatment using a F rench press or shaker. Subsequently, the liquid containing the disintegrated cell body is centrifuged to obtain a supernatant liquid containing no substantial lipopolysaccharide endotoxin and Oantigen. The thus obtained supernatant liquid is purified according to chromatography using as the adsorbent DEAE- cellulose (diethylaminoethyl cellulose), Sephadex (a trademark for a dry insoluble powder composed of microscopic beads which are synthetic organic compounds derived from the polysaccharide dextran, manufactured and sold by Pharmacia Fine Chemicals, Inc., New Jersey, U.S.A.), or DEAE-Sephadex, whereby a desired effective fraction can be isolated. 3. The aboven egtjgne d lhase 111, Phase II or Phase I organism of Bordetella pertusis is cultured at 35 to 37C. for 2 to 3 days in any one of a solid culture medium suitable for culturing Bordete l la pertusis such as a charcoal medium, and the resulting cell body is treated inthe same manner as above, whereby an effective fraction can be isolated as well.

Procedures for producing interferon inducers according to the present process are illustrated below with reference to examples, but the examples are merely illustrative and do not limit the scope of the present invention.

EXAMPLE 1 A Phase [II organism of Bordetella pertusis was subjected to shaking culture at 35 to 37C. for 72 hours in a modified Cohen-Wheeler culture medium. After the cultivation, the cultured liquid was centrifuged to obtain 1,000 ml. of a supernatant liquid. This supernatant liquid was filtered under sterile conditions, whereby 900 ml. of an effective fraction (A) was obtained.

In the same manner as in the case of the Phase III organism 900 ml. of an effective fraction (B) was obtained by use of a Phase II organism of Bordete/Ia pertusis. i

In the same manner as in the case of the Phase III organisrr 900 m1. 'of an effective fraction (C) was obtained by nears Page I orglanisinof B'Ordaaua pertusis.

The above-mentioned effective fraction (A) was concentrated to 50 ml. then this concentrate was purified according to chromatography using DEAE-Sephadex A-50 to obtain 50 ml. of an effective fraction. Then thus obtained effective fraction is purified similarly using Sephadex 6-50 to obtain 50 ml. of another effective fraction.

EXAMPLE 2 Al hase IIT organism of Bordelella pert us i's was cu] tured at 35 to 37C. for 48 hours in a charcoalagar cul ture medium [J.R. Norris and D.W. Ribbons: Methods in Microbiology, pp. 4, 97 and 283 (1970), Published from Academic Press Inc., London-New York]. Thereafter, 10 g. of the cell body collected was suspended in 100 ml. of a phosphate buffered-saline solution, and the resulting cell suspension was subjected for 30 minutes to 10 kc. sonic treatment to disintegrate the cell body. Subsequently, the liquid containing the disintegrated cell body was centrifuged to recover ml. of a supernatant liquid. This supernatant liquid was treated according to chromatography using Sephadex G 150, whereby 40 ml. of an effective fraction (D-l) was obtained.

In the same manner as in the above, the Phase [11 organism of Bordetella pertusis was cultured at 35 to 37+C. for 72 hours, whereby 40 ml. of an effective fraction (D-2) was obtained.

in the same manner as in the case of the Phase III organism, 40 ml. of an effective fraction (E) was obtained by use of a Phase 11 organism of Bordete lln pertusis.

In the same manner as in the case of the Phase [11 organism, 40 ml. of an effective fraction (F) was obtained by use of a Phase 1 organism of Bordetella pertusis. Further, the liquid containing the disintegrated cell body of the Phase I organism was centrifuged to obtain a supernatant liquid, which was then subjected to chromatography using DEAE-cellulose as the adsorbent, whereby 30 ml. of an effective fraction (G) was obtained.

The results of determination of interferon production of the effective fractions (A) to (G) obtained in Examples 1 and 2 are mentioned below with reference to the following reference examples.

REFERENCE EXAMPLE 1 Determination of production amounts of interferon (in vitro test):

A mixed solution comprising 1.4 parts by volume of a phosphate buffered-saline solution and 1 part by volume of Ringers solution was mixed with 0.6 part by volume of a calf serum, and the resulting mixture was charged with 3 parts by volume of distilled water to prepare a hypotonic solution. Using this hypotonic solution, each of the effective fractions (A) to (G) was diluted to various concentrations to prepare diluted samples. On the other hand, a cell suspension was prepared in such a manner that a rabbit of 500 to 1,000 g. in body weight was sacrificed by cardiac puncture, the spleen and lymph node thereof were recovered, the tissues of said organs were torn with forceps to disperse cells, and the cells thus obtained were suspended in a culture medium to a concentration of 5 X cells/dish for spleen and lymph node. Subsequently, each of the aforesaid diluted samples was mixed with the abovementioned cell suspension and cultured at C. for 24 hours, and the cultured liquid was centrifuged to obtain a supernatant liquid, which was then subjected to determination of interferon production. The results obtained were as set forth in Table 1.

(The titer of interferon was expressed in terms of the highest dilution causing a percent inhibition of the number and size of plaques in comparison with untreated controls.)

Table 1 Determination of interferon production (in vitro test) Titer of interferon Concentration Sample of sample Spleen cell Lymph node cell 1. Phase [11 organism ofBordet/Ia perrusis (1) Supernatant liquid 10 X dilution of cultured medium [Effective do. 220 700 fraction (A)] 1,000 do. 220 680 (2) Supernatant liquid ofdisintegrated cell body (48 hours culture) [Effective fraction (D-1)| 100 X dilution 430 330 1,000 do. 340 310 10,000 do. 230 330 100,000 do. tc 100 (2) Supernatant liquid of disintegrated cell body (72 hours culture) [Effective fraction (D2)] 100 X dilution 500 1,000 do. 36. 230 10,000 do. 205 220 IO0,000 do. 50 85 2. Phase ll organism of Br irdelella porruxis (I) Supernatant liquid 100 X dilution 280 of cultured medium (72 hours 1,000 do. 220

culture) [Effective fraction (8)] (2) Supernatant liquid 100 X dilution 320 of disintegrated cell body (48 1,000 do. 300

hours culture) [Effective fraction (E)] Determination of interferon production (in vitro test) Table l 4 Continued Titer of interferon Concentration Sample of sample Spleen cell Lymph node cell 3. Phase 1 organism of Bordetella pertusis (l) Supernatantliquid X dilution I06 17 of cultured medium [Effective 100 do. 320 52 fraction (C)] 500 do. 160 72 (2) Supernatant liquid Containing 10 pg. 930 240 of disintegrated of solid per ml.

cell body (untreated) [Effective do. fraction (F)] (treated at 56C.

for minutes) 1,400

(3) Necrotoxin l0 Times-dilution of liquid contain- 720 L900 [Effective ing 45 ug of solid fraction (0)] per ml.

50 Times-dilution 630 1,900

of said liquid 250 Times-dilution 230 of said liquid not conducted) REFERENCE EXAMPLE 2 Determination of interferon production (in vivo test):

Each of the diluted samples prepared in Reference 30 Example 1 was administered in a does of 0.5 ml. by intravenous injection to a rabbit of 500 to 1,000 g. in body weight. At the 2nd and Sthe hours after the injection, each 3 ml. of the blood of the rabbit was taken up by cardiac puncture, and the serum isolated from said blood was subjected to determination of interferon production in the same manner as in Reference Example I. The results obtained were as set forth in Table 2.

Table 2 Determination of interferon production (in vivo test) for 30 minutes) Sample Concentration Rabbit of sample No. After 2 hrs. After 5 hrs.

1. Phase III organism of Bordetella perms-is Supernatant liquid l0 X dilution 7 490 50 of cultured medium [Effective do. 8 I800 2 I 0 fraction (A)] 2. Phase I organism of Brlrdetella perlusis (l) Supernatant liquid l0 X dilution 1 600 of cultured medium (Effective do. 2 2200 I30 fraction (C)] do. 3 2200 2H) (ll Supernatant Containing 10 pg 7 liquid of of solid per ml. 4 1200 40 disintegrated untreated cell body do. [Effective fraction (F)] (treated at 56C. 5 1250 for 30 minutes) (3) Necrotoxin 500 aglkgwt. 6 340 40 (untreated) [Effective d fraction G 0.

( )1 (treated at 56C. 9 43 less than I0 not conducted) As is clear from the results shown in Tables 1 and 2, it was confirmed that the supematants of cultured media and disintegrated cell bodies of the Phase I, ll and l organisms of Bordetella pertusis produced interferons both in vitro and in vivo tests using rabbits. Particurly, it wassqll srysdrthauhgsmaema nt Obtained from the Phase III organism of Bordetella pertusis were lowest in toxicity and excellent in effect of inducing the interferon production;

Further, it was confirmed that virus-inhibiting factors induced by the interferon inducers obtained according to the present process were not effective on chicken embryo cells different in species, had actions of inhibiting the propagation of visicular stomatites virus and vaccinia .virus on lined cell RK. 13, and had such characteristics of interferons that they were inactivated with 0.08 trypsin at 37C., did not precipitate even when centrifuged with a centrifugal force of 100,000 g.

' and were not dialyzed with a cellophane bag.

What is claimed is:

l. A process for the preparation of an interferon inducer for viral infections which comprises culturing phase Tl or lll Brdtlla per i u'sis iii a liquid cul ture medium, separating a supernatant liquid from the cultured medium, filtering the thus obtained supernatant under sterile condition to obtain a filtrate, extracting the thus obtained filtrate by ultra filtra- 8 tion or chromatography to obtain the effective fraction, said effective fraction being capable of providing an interferon inducing effect.

2. An interferon inducer effective for viral infections comprising an effective fraction of Fbfdtella p e iiitiis produced by culturing Bordetella pertusis in a liquid culture medium, separating a supernatant liquid from the cultured medium, filtering the thus obtained supernatant under sterile conditions to obtain a filtrate, extracting the thus obtained filtrate by ultra filtration or chromatography to obtain said effective fraction virus inhibiting factors induced by said effective fraction having the characteristics of (i) not affecting the chicken embryo cells in different species; (ii) inhibiting the propagation of visicular stomatities virus and of vaccinia virus on lined cell RK-l3; (iii) being inactivated with 0.08% trypsin at 37C; (iv) not being precipitated even when centrifuged with a centrifugal force of 100,000 g.; and (v) not being dialyzed with a gellophane bag, the BordeteI/a pertusis being a phase Ill or phase II organism of Bordetella pertusis.

3. An interferon inducer according to claim 2 wherein the Bordetella pertusis is in phase III.

4. A process according to claim 1, wherein Bordetella pertusis is cultured in shaking culture by using a modified Cohen Wheeler liquid medium at 35? to 37 C for 24 to 72 hours. 

1. A PROCESS FOR THE PREPARATION OF AN INTERFERON INDUCER FOR VIRAL INFECTIONS WHICH COMPRISES CULTURING PHASE II OR III BORDETALLA PERTUSIS IN A LIQUID CULTURE MEDIUM, SEPARATING A SUPERNATANT LIQUID FROM THE CULTURED MEDIUM, FILTERING THE THUS OBTAINED SUPERNATANT UNDER STERILE CONDITION TO OBTAIN A FILTRATE EXTRACTING THE THUS OBTAINED FILTRATE BY ULTRA FILTRATION OR CHROMATOGRAPHY TO OBTAIN THE EFFECTIVE FRACTION, SAID EFFECTIVE FRACTION BEING CAPABLE OF PROVIDING AN INTERFERON INDUCING EFFECT.
 2. An interferon inducer effective for viral infections comprising an effective fraction of Bordetella pertusis produced by culturing Bordetella pertusis in a liquid culture medium, separating a supernatant liquid from the cultured medium, filtering the thus obtained supernatant under sterile conditions to obtain a filtrate, extracting the thus obtained filtrate by ultra filtration or chromatography to obtain said effective fraction virus inhibiting factors induced by said effective fraction having the characteristics of (i) not affecting the chicken embryo cells in different species; (ii) inhibiting the propagation of visicular stomatities virus and of vaccinia virus on lined cell RK-13; (iii) being inactivated with 0.08% trypsin at 37*C; (iv) not being precipitated even when centrifuged with a centrifugal force of 100,000 g.; and (v) not being dialyzed with a cellophane bag, the Bordetella pertusis being a phase III or phase II organism of Bordetella pertusis.
 3. An interferon inducer according to claim 2 wherein the Bordetella pertusis is in phase III.
 4. A process according to claim 1, wherein Bordetella pertusis is cultured in shaking culture by using a modified Cohen -Wheeler liquid medium at 35* to 37* C for 24 to 72 hours. 